Vertical transmission of chikungunya virus in Aedes albopictus Michele Dottori1, Romeo Bellini2, Anna Medici2,Francesca Cavrini3, Mattia Calzolari1, Vittorio Sambri3 ,Paolo Bonilauri1, Paolo Cordioli1, Paola Angelini4. 1 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia‐Romagna “B. Ubertini”, 2 Brescia, Italy Centro Agricoltura Ambiente “G.Nicoli”, Settore Entomologia Medica e Veterinaria, Crevalcore, Italy 3 Centro di Riferimento Regionale per le Emergenze Microbiologiche, Bologna, Italy 4 Regione Emilia‐Romagna, DG Sanità e Politiche Sociali, Bologna, Italy Emerging vectorborne disease: the role of Aedes mosquitoes Cervia (Ravenna, Italy) May 9-10, 2011 The 2007 outbreak of CHIKV in Italy • About 250 diagnosed cases, mainly in Castiglione di Ravenna and Castiglione di Cervia. • First report of autochthonous cases of CHIK outside Africa and Asia. 2 The vector: Asian tiger mosquito • Asian tiger mosquito (Aedes albopictus): an exotic species of mosquito now very common in Italy. • A great risk for the overwintering of virus was the vertical transmission from mosquito mothers to their offspring. foto CAA 3 Experiment on vertical transmission Human Human strain strain 7.3 TCID /ml A1=10 A1=107.3 TCID50 50/ml 6.3 TCID A2=10 /ml A2=106.3 TCID50 50/ml 5.3 TCID A3=10 /ml A3=105.3 TCID50 50/ml Mosquito Mosquito strain strain 5.5 B1=10 /ml TCID50 B1=105.5 TCID 50/ml 5.0 TCID B2=10 /ml B2=105.0 TCID50 50/ml 4.5TCID B3=10 /ml B3=104.5 TCID50 50/ml Controls Controls 4 Experiment on vertical transmission • The oral infection was conducted in BLS3 in 12 Plexiglas cages (18x18x18 cm ) with 60 females/cage, 4-5 days old • 6 blood infected meals (A1, A2, A3, B1, B2, B3) • 2 blood meals consisting of human washed erythrocytes • 2 with washed erythrocytes mixed with the culture medium used for the virus suspension •2 with mechanically defibrinated rabbit blood, • In all cases, immediately after the blood feeding session, the non-engorged females were removed from the cages and eliminated. 5 Results (1): egg fertility • Significant reduction in the fertility of eggs laid by females blood fed with the virus suspension, when compared with blood meal without virus . Cage N of Fertility cages A-Rabbit blood 2 92.1 B-Human erythrocytes 2 89 C-H. erythrocytes+DMEM 2 90.5 D-H. erythrocytes+virus A 3 11.6 E-H. erythrocytes+virus B 3 11.9 Fertility: percentage of hatched eggs Data referred to the first blood meal 6 Results (2): mosquito mothers • Legs and body of mosquito mothers were separately tested: positive legs were considered evidence of the diffusion of the virus in the insect. • A total of 84 engorged females were tested. • Complete virus diffusion in A1, B1, B2. DPI 6 14 19 Cage s b/l s b/l s b/l A1 3 3/1 3 2/1 10 10/10 A2 3 3/2 3 1/0 4 0/0 A3 3 1/1 3 0/0 13 3/1 B1 3 3/1 2 2/2 2 2/2 B2 3 2/0 3 3/3 3 3/3 B3 3 2/2 3 1/1 3 2/2 DPI=days post infection s=sampled b=body 7 l=legs Results (3): offspring • Total progeny tested: 689 adults • Positive: 2 males (from cage B2 and B3) and 1 female (from cage A1), all stemming from the second gonotrophic cycle. • Considering only the total female progeny of cage A1, 0.88% (1/113) is the upper limit of vertical transmission of CHIKV to female progeny detected in this experiment. male female Total A1 129 113 242 A2 35 38 73 A3 106 81 187 B1 32 27 59 B2 57 49 106 B3 12 10 22 Total 371 318 689 8 Conclusions • The highest concentrations of CHIKV strains utilized infected all the mosquito mothers at the end of experiment. • The virus reduces fertility of eggs • The positive insects in the offspring are all detected in the second gonothopic cycle. • CHIKV infected mosquito females are capable of transmitting the virus vertically to their female progeny at a maximum rate below 1% (infective virus?). • The rate of vertical transmission of CHIKV in Tiger mosquitoes isn’t probably sufficient to guarantee the maintenance of the virus in a temperate region. 9